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allergic alveolitis in a library. Em J Respir Dis 1982;63(suppl 124):32. 19. Fergusson RJ, Mime LJR, Crompton GK. Penicilliurr-lllergic alveolitis: faulty installation of central heating. Thorax 1984;

39:294. 20.

Hoffman extracts.

DR, Kozak PP. Shared and specific allergens J ALLERGY CL~ IMMUNOL 1979;63:213.

in mold

21.

Salvaggio J, Aukrust L. Mold-induced asthma. J ALLERGY CLIN IMMUNOL 1981;68:327. 22. Sosman AJ, Schlueter DP, Fink JN, Barboriak JJ. Hypersensitivity to wood dust. N Engl J Med 1969;281:977. 23. Howie AD, Boyd G, Moran F. Pulmonary hypersensitivity to Ramin (Gonysrylus bancunus). Thorax 1976;3 1585.

K channels of human alveolar macrophages Yasunori Kakuta, MD, Hiroshi Okayama, MD, Takashi Aikawa, MD, Tomohiko Kanno, MD, Tadasu Ohyama, MD, Hidetada Sasaki, MD, Taizo Kato, MD,* and Tamotsu Takishima, MD Sendui, Japan The activation of macrophages has been reported to be associated with Ca-activated K permeability change. In order to study this permeability change in human alveolar macrophages, we examined alveolar macrophages electrophysiologically at a single channel level. We observed two types of Ca-activated K channel currents having conductances of 218 2 2 and 32 ? 0.6 picosiemens in symmetrical 154 mmollL KC1 solutions. The characteristics, such as voltage dependency and Ca sensitivity, as well as channel conductance, were dtjferent between these two types of channel currents. Quinine (a blocker of Ca-activated K conductance), 0.5 mmollL, reduced these channel currents by 45 2 8% and 31 ? 8%. Quinine, 0.5 mmollL, also inhibited chemiluminescence and leukotriene B, release by 82 + 6 to 88 +- 3% and 88 ? 2%. respectively. These results suggest the presence of two types of Ca-activated K channels, which may be related to the relase of inflammatory mediators from human alveolar macrophages. (J ALLERGY CLIN IMMUNOL 1988;81:460-8)

Alveolar macrophages possess Fc receptors for IgE,’ and stimulation with IgE release various mediators, including superoxide anion,* which affects epithelial-barrier function and increases the permeability of the paracellular pathway.3 This effect of alveolar macrophages together with chemical mediators from mast cells in the airways may allow the antigen to penetrate into and induce further allergic reactions in the bronchial tissues.4 The increase in the permeability may also facilitate nonspecific stimulants reaching bronchial irritant receptors, causing increased sensitivity of the bronchi.’ Alveolar macrophages, stimulated with such agents as endotoxin, From the First Department of Internal Medicine and *Department of Dermatology, Tohoku University School of Medicine, Sendai, Japan. Supported in part by grants from the Ministry of Education, Science, and Culture, Japan. Received for publication June 27, 1986. Accepted for publication Aug. 22, 1987. Reprint requests: Tamotsu Takishima, MD, Professor and Chairman, First Department of Internal Medicine, Tohoku University School of Medicine, l-l Seiryo-machi, Sendai, 980, Japan.

460

Abbreviations

used

LTB,, LTC,: PMA: ZAS: HPLC:

Leukotrienes B, and C, Phorbol myristate acetate Zymosan activated serum High-performance liquid chromatog raphy Intracellular-free Ca concentration Picosiemens N-2-hydroxyethyl-piperazine-N’-2ethanesulfonic acid Calcium

[Cal,: pS: HEPES: Ca:

lymphokines, or immune complexes, release plateletactivating factor,6 LTC,,’ and LTD,, which may be important mediators of bronchoconstriction.*~ 9 Specific allergens cause alveolar macrophages from patients with asthma to release a chemotactic factor for neutrophils and eosinophils,” which, particularly eosinophils, gather in the bronchoalveolar space during the late asthmatic reaction” and cause epithelial damage. ‘* Furthermore, macrophages may cause basophils

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to release mediators during the late-phase reaction. I3 Therefore, alveolar macrophages appear to play a significant role in asthma. This interpretation of the role of alveolar macrophages in asthma suggests that the regulation of the activation of alveolar macrophages could be a useful step in the management of asthma. However, the activation process in stimulation of macrophages is unclear. In mouse peritoneal macrophages and human monocyte-derived macrophages, membrane hyperpolarization has been observed after stimulation with endotoxin-activated serum, N-formyl-methionylleucyl-phenylalanine, and Ca iopophore A23 187. “, I5 The hyperpolarization is caused by change in K permeability that is in turn regulated through intracellular Ca (i.e., Ca-activated K conductance). I6 Although the role of this permeability change is not clearly understood,” modification of macrophage activation by K has been recently postulated.” In order to study the Ca-activated K permeability change in human alveolar macrophages, we used an electrophysiologic method (patch-clamp techniques)lR to determine whether such a mechanism is present in human alveolar macrophages. The high resolution of the patch-clamp method made it possible to examine K permeability changes at the single channel level. Then, we examined the effect of quinine on single channel currents because quinine has been reported to inhibit Ca-activated K permeability change in mouse peritoneal macrophages.” Finally, we studied the effect of quinine on lucigenin-dependent chemiluminescence response and LTB, release of human alveolar macrophages to study whether reduction of channel currents is associated with inhibition of inflammatory mediator release. Quinine has been reported to reduce superoxide anion release from guinea pig alveolar macrophages.‘” The chemiluminesence method is very sensitive for observing activation of phagocytic cells.” LTB, is one of the crucial mediators of asthma.2” Some preliminary studies have been presented elsewhere.” 24 METHODS Human alveolar macrophageswere obtained from bronchoalveolar lavage of 23 patients (six smokers and 17 nonsmokers) who were undergoing diagnostic fiberoptic bronchoscopy for bloody sputum of unknown origin, solitary tumor, or exclusion of sarcoidosis. Informed consent was obtained from all patients studied. The preparation of samples is described elsewhere.25. 26Briefly, cells adhering to a small piece of cover glass were transferred to a dish of l-ml capacity, in which macrophages were identified by the criteria of Saltini et al.,*’ with a phase contrast or a plain inverted microscope at a magnification of x600. Initial viability of macrophages was >95%, as judged by exclusion

K channels

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of 0.1% trypan blue, typically decrcasmg a!ightiy :o X851 by the end of the 2-hour experimentation pc! toe,

Patch-clampamplifiers (EPC-7, 1.~ Elecrron~cs,Darrn stadt, Federal Republic of Germany) were usual mr the measurement of single channel currents, Data wcrc filtered with a 3 kHz low-pass filter and recorded with a~ FM tape recorded (0 to 2 kHz frequency response). ‘f’t~s~ data were filtered through a Bessel low-pass hlter (bu~:r 1-1our laboratory) at 1 to 2 kHz and fed into a compute1 (7 1’17, NECSan-ei, Tokyo, Japan) at a sampling intervai or 0.2 to 0.5 msec for analysis, according to the method of Barrett et al.” Voltages are described as follows. nc+itive voltage refers to internal surface negative t<~ eicternzl surface (hyperpolarization), and positive voltage refcrr !i) internnl \urface positive to external surface (depolanzai’:oni To study single ion channels, standard patch-clamp techniques” were used. Briefly, an eicctn*dr ircsi~tancc 2 to 4 M a) was placed onto the surtace ~~~entbranc of a macrophage. and a high resistance seal iL tl: 20 G fl) was established by applying slight suction throueh the pipette. The electrode with attached membrane W:I” then removed from the cell, and either “inside-out” or ‘“~~u:~&~-ou~” configurations were formed. In the former cast. I.ZXC’ !ntracellular surface of the membrane faces the bathmg solirti
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FIG. 1. Single channel currents obtained from human alveolar macrophages and effect of membrane potential on channel activity of excised “inside-out” membrane patch. The experiment was performed in symmetrical 154 mmol/L KCI solutions. Ca concentration perfusing the intracellular surface [Cali was 3 pmol/L. A, Large single channel currents at various membrane potentials. Upward-going currents imply currents outward from the internal surface to the external surface of the membrane; p/l indicates picoampere = lo-‘*A, o indicates channel opening and, c indicates that the channel is in the closed state. Ca concentration of the solution at the external surface of the membrane [Calo, was ~~10 nM. The channel currents are displayed after Bessel-type filtering with a high frequency cutoff at 500 Hz. B, Small channel currents at various voltages; o, to o3 indicates simultaneous opening of 1 to 3 channels. [CaJo was 2 mmol/L. C, Percent open dwell time as a function of membrane potential; to), large channels; (A), small channels. Each value is the mean of five to seven experiments. Vertical bar indicates -C 1 SEM. [Calo was
anesulfonate). In some experiments, KC1 of the solution was replaced with other ions, as described in Results. Experiments were performed at room temperature (20” to 23” C). Chemiluminescence was measured, as described previously,“’ except that lucigenin was used instead of luminol. Briefly, the coverslip with attached alveolar macrophages

(1 to 2 x lo5 cells) was incubated in 1 ml of Dulbecco’s modified Eagle’s medium buffered with 25 mmol/L of HEPES (pH 7.4) at 37” C, containing 0.2 mmol/L of lucigenin (Sigma Chemical Co.). Background chemiluminescence was measured with a Biolumat (LB9500, Berthold, Wildbad, Federal Republic of Germany). Quinine was added after 5 minutes in some experiments. In control experiments, phosphate-buffered saline without the divalent cation was added at this time. After another 5-minute incubation, macrophages were stimulated with PMA (final concentration, 0.5 pg/m1)j2 or ZAS (final concentration lO%).j’ Chemiluminescence was measured and expressed as a percent peak response of control experiment . The effect of quinine on LTB, release was examined according to the method of Williams et al.,” with slight modification, by use of reverse-phase HPLC. Briefly, culture dishes, including 1 to 2 X lo6 alveolar macrophages, were exposed to 2 pmol/L of Ca ionophore A23 187 (Calbiochem-Behring Corp., La Jolla, Calif.) for 30 minutes in Tyrode’s solution with 25 mmol/L of HEPES at pH 7.4. In some cases, quinine was applied 5 minutes before exposure of Ca ionophore A23 187. The samples were passed through a cartridge of octadecylsilyl silica (SEP-PAK, Cl8 cartridge; Waters Associates, Millford, Mass.) and were then separated with HPLC on a Cl&NOVA PAK cartridge (3.9 X 150 mm; Waters Associates). Sequential elution was carried out with methanol/water/triethylamine, 67 : 32.5 : 0.5 (vol/vol/vol), adjusted to pH 5.2 with acetic acid at a flow rate of 0.5 ml/min. LTB, was quantified by comparing the areas of the absorbance at 280 nm with that of the synthesized LTB, (a gift from Takeda Chemical Industries, Japan). The amount of LTB, released in the presence of quinine was compared with that in the absence of quinine. Preliminary radioimmunoassays (Amersham, England) and chemotaxis assays were consistent with the separation of LTB, in our column effluents. The amount of cellular protein was determined by Lowry method.34

RESULTS The single channel currents observed in excised “inside-out” patch membrane under different voltages are illustrated in Fig. 1. By depolarization of the membrane potential to 40, 60, and 80 mV, the outward channel currents with relatively large amplitudes are observed in Fig. 1, A. The channel currents spend more in the open state with increasing membrane potential in Fig. 1, A. When the membrane potential was hyperpolarized, the channel currents with large amplitudes were barely observed, especially at low (3 pmol/L and less) Ca concentration on the cytoplasmic surface of the membrane ([Cal,) (comparison of percent open dwell time at - 20 mV with those at 60 or 80 mV, Fig. 1, C). In such conditions as hyper-polarization (- 100 to -40 mV) and low (1 to 3 pmol/L) [Cal,, the inward channel currents with relatively small amplitudes were clearly observed

K channels

VOLUME E? NUMBER 2

current

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FIG. 2. The single channel current amplitude as a function of out” configuration was used. Outward currents are plotted as nM and 10 pmol/L for large channel currents, and 2 mmol/L currents, respectively; (0) and (A) symmetrical 154 mmol/L channels, respectively. Large (0) and small (A) channel current mmol/L NaCl plus 54 mmol/L KCI at extracellular surface mmol/L KCI solution at intracellular surface are also illustrated. to 10 experiments, and vertical bars are t 1 SEM, but values smaller than the size of the symbols. The curves were fitted square method, except the curve of (e), which is fitted to the Katz K flux equations.@

without the interference of the channel currents with large amplitudes (Fig. 1, B). The percent of open dwell time of the channels with small amplitudes did not appreciably change at membrane potentials from - 40 to - 100 mV (Fig. 1, C). The channel conductances in symmetrical 154 mmol/L KC1 solutions were 218 2 pS (lo-‘* Sz-‘) (n = 40 cells) for the channel currents with large amplitudes ( - 50 to 50 mV) and 32 t 0.6 pS (n = 35 cells) for the channel currents with small amplitudes ( - 90 to - 40 mV). The conductances did not appreciably change with either external or internal Ca concentration (data not presented). Therefore, we conclude that there are at least two types of channel currents with different voltage dependency and conductances in the membrane of human alveolar macrophages. We call these channel currents large and small channel currents for convenience To determine the ion selectivity of the channels, we examined the effect of ion substitution on channel currents. The results are illustrated in Fig. 2. In symmetrical 154 mmol/L KC1 solutions with no K or Cl gradient between both sides of the membrane (154 mmol/L KC1 outside per 154 mmol/L KC1 cytoplasmic side), the reversal potentials (0 current potentials) of large and small channel currents were estimated to

of aiveol,r:

membrane potential. The “insidepositive. [Ca]o and [Cal, were < 10 and 1 kmol/L for small channel KC1 solution for large and small amplitudes in a condition of 100 of patch membrane, and 154 Each value is the mean of five are not presented when they are to the data points by the least data points by Goldman-Hodgkin-

be - 0.5 and - 3.1 mV, respectively (Fig. 2). Since the K or Cl equilibrium potential without gradient is 0 mV, the estimated value was very close to K or Cl equilibrium potential. Extracellular K was then reduced by substitution with Na, 100 mmol/L (100 mmol/L NaCl plus 54 mmol/L KC1 outside per 154 mmol/L KC1 cytoplasmic side). The estimated reversal potentials of large and small channel currents shifted to - 26.8 and - 25.9 mV, respectively ( Fig. 2). The K equilibrium potential in this condition is - 26.5 mV caused by the K gradient, whereas the Cl equilibrium potential is still 0 mV m the absence of a Cl gradient. Therefore, the values, - 26.8 and - 25.9 mV, were comparable with the K equilibrium potential. Moreover, these values were far from the Na equilibrium potential (>290 mV, with 100 rnmol/L Na outside and assuming leaked Na concentration at cytoplasmic side to be < 1 pmol!L). When extracellular Cl was largely replaced by an equimolar concentration of methanesulfonate (150 mmol/L K-methanesulfonate plus 4 mmol/L KC1 outside per 154 mmol/L KC1 cytoplasmic side), the estimated reversal potentials of large and small channel currents were - 3.3 and - 6.0 mV, respectively. These values were still close to the K equilibrium potential of 0 mV compared with the Cl equilibrium potential of 92 mV.

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FIG. 3. Effect of [Cal, on channel activity of excised “insideout” membrane patch. Symmetrical 154 mmol/L KCI solutions were used. A, Tracings of large channel currents with various [Cali; 0 indicates channel opening; C indicates that the channel is in the closed state. Membrane potential was +20 mV. [[Calo was
Therefore, these results indicate that both large and small channel currents are strongly selective to K compared with Na or Cl. We next examined the effect of K and Na channel blockers to confirm further the ion selectivity of the large and small channel currents. Tetraethylammonium, which is known to block K channels,35 reduced the conductances of large and small channel currents to 83 & 9 pS (n = 5 cells) and 23 + 1 pS (n = 5 cells), respectively, by intracellular application at a concentration of 60 mmol/L. In contrast, tetrodotoxin, 1 Fmol/L, which maximally blocks reconstituted fast Na channels and reduces open probability,36 did not noticeably affect the activities of large or small chan-

Sfriall channel

0.2

Quinine

0.5

(mid)

FIG. 4. Effect of quinine on large and small channel currents of excised “outside-out” membrane patch. Experiments of large channel currents were performed in 150 mmol/L NaCl plus 4 mmol/L KCI solution at external surface of the membrane and 154 mmol/L KCI solution at internal surface of the membrane. Symmetrical 154 mmol/L KCI solutions were used in small channel experiments. [Calo was 2 mmol/L. A, a: Traces of large channel currents with no quinine (control) or 0.5 mmol/L of quinine at a membrane potential of +60 mV. [Cal, was 0.1 Fmol/L. The channel currents are displayed after Besseltype filtering with a high frequency cutoff at 1 kHz. A, b: Peak average current during a voltage step from - 10 to +60 mV in the presence or absence of quinine. [CaJ was 1 pmol/L. Channel currents were displayed through a high frequency cutoff at 1 kHz. B, a: Traces of small channel currents with no quinine (control] or 0.5 mmol/L of quinine at -60 mV. [Cal, was 3 PmollL. Channel currents were displayed through a high frequency cutoff at 2 kHz. B, b: Same traces as illustrated in B. a, displayed in slower time scale. Channel currents were displayed through a high frequency cutoff at 500 Hz. C, Dose response of quinine on large and small channel currents. Vertical bar indicates -cl SEM (n = 5); p values refer to significant differences from control. *p < 0.05; **p < 0.01.

nel currents (data not presented). These results were also consistent with the idea that large and small channel currents flow through K channels. The effect of Ca at cytoplasmic side ([Cal,) on channel activities was examined to determine the degree

VOLUME a1 h!lJMRER 2

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QUININE(mM)

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@ Aa L 0

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of Ca activation. In Fig. 3, A, the effect of [Cal, on the large channel currents is illustrated. At 0.1 pmol/L [Cal,, the large channel currents are almost in the closed state, and very brief transitions to open state are observed. By increasing [Cal,, the large channel currents tend to shift to the open state, and at 100 pmol/L [Cal,, the large channel currents are almost fully activated (Fig. 3, A). The effect of [Cali on small channel currents is illustrated in Fig. 3, B. At low [Cali (0.3 FmoliL), the small channel currents are seldom observed, but when [Cal, is increased (3 Fmol/L and more), small channel currents frequently shift to the open state. Small channel currents cannot be fully activated even at 100 pmol/L [Cal, in this figure. The difference of the Ca sensitivity of large and small channels is further illustrated in Fig. 3, C, which demonstrates the increase in the percent open dwell time of large channel currents with increasing [Cali. The percent open dwell time of small channel currents were also markedly increased at 10 Fmol/L [Cal, and above as compared with that at 0.3 pmol/L, but the small channel currents were not fully activated even at 100 pmol/L [Cal, (Fig. 3, c). Therefore, both large and small channel currents appear to be Ca-activated K channel currents, but with different sensitivities to Ca. The effects of quinine on large and small channel currents are illustrated in Fig. 4. In Fig. 4, A, with

i) 5

0.2

0.1

(mM1

FIG. 6. Effect of quinine on the LTB, release stimulated with Ca ionophore A23187 at 2 kmol/L for 30 minutes. A, Reverse-phase HPLC profiles of metabolites released from human alveolar macrophages. The ultraviolet absorbance profile was monitored at 280 nm. An arrow of each panel corresponds to the elution time for the synthetic standard of LTB,. Large peak of right panel at the elution times of 4 to 8 minutes are related to quinine itseif, because quinine alone demonstrated the same profile idata not presented). B, Dose-response effect of quinine on the LTB, release. Values are expressed as percent of control experiments (Ca ionophore alone). Vertical bar indicates 2 1 SEM (n = 5); p values refer to significant differences from control. **p < 0.01.

application of 0.5 mmol/L of quinine, large channel currents were interrupted brietly and lost the characteristic rectangular shape. Since these interruptions were too brief to resolve as individual events: we examined the effect of quinine on the average channel currents obtained during a voltage step from -- 20 mV to 60 mV, as illustrated in Fig. 4, A and R. We compared the peak average current, and the results are summarized in Fig. 4, C. The peak cm-rents were reduced by 45% in the presence of 0.5 mmolif, of quinine. We next examined the effect ot’ quinine on the small channel currents, which is Illustrated in Fig. 4, B in different two-time scale. r>uinine. 0.5

466

Kakuta et al.

mmol/L, also blocked the small channel currents, appearing as if quinine increased the closed channel state. The percent open dwell time of small channel currents in the presence of 0.5 mmol/L of quinine was decreased by 31% (Fig. 4, C). These results indicate that quinine inhibits both large and small channel currents, but with different mode of action. We examined the effect of quinine on the chemiluminescence response induced by PMA and ZAS. As illustrated in Fig. 5, quinine, at 0.2 to 0.5 mmol/L, inhibited the chemiluminescence response stimulated with these compounds. Cell viability was not decreased by 0.5 mmol/L of quinine, being 98 ? 3% (n = 5) of control, which was >95%. We examined the effect of quinine on LTB, release. In Fig. 6, A, (left panel) the representative reversephase HPLC profiles of metabolites released from human alveolar macrophages are illustrated. The relative recovery of LTB4, standard from our experimental system, was 83 + 3% (n = 5), and we estimated the yield of LTB, as 202 -+ 20 pmol/lOO pg of cell protein, comparable with Fels et a1.37 Quinine, 0.5 mmol/L, reduced the LTB, release (Fig. 6, A, right panel). The inhibitory effect of quinine on LTB, release is summarized in Fig. 6, B. As illustrated in this figure, quinine, 0.2 and 0.5 mmol/L, inhibited LTB, release. Quinine, 0.5 mmol/L, however, did not noticeably change relative recovery of LTB, standard (81 + 4%, n = 5; compared with control) nor the viability of macrophages (98 + 2% of control [>95%], n = 5). Quinine inhibited both the chemiluminescence response and LTB, release. In separate experiments, quinine inhibited both large and small Ca-activated K channel currents in human alveolar macrophages. Therefore, inhibition of inflammatory mediator release by quinine was closely associated with K channel blockade in human alveolar macrophages. DISCUSSION

We observed two (large and small) types of single channel currents in excised membrane patches of human alveolar macrophages. Both channel currents were selective to K (Fig. 2) with channel conductantes reduced by tetraethylammonium and were unaffected by tetrodotoxin. The activity of both channel currents were increased with increasing [Cal, ( Fig. 3). These data are consistent with the idea that the channel currents flow through Ca-activated K channels. There were, however, some different characteristics between large and small channel currents, as follows: Principally, the unit conductances were different (218 + 2 pS, large channels; 32 + 0.6 pS, small channels). Second, voltage dependency of channel

J. ALLERGY CLIN. IMMUNOL. FEBRUARY 1988

opening was not similar to each other (Fig. 1). Third, large and small channel currents demonstrated a different Ca sensitivity (Fig. 3). Finally, quinine blocked these channel currents in different ways (Fig. 4). These results are consistent that large and small channel currents flow through two different types of channels. The properties of large channel currents resemble those of Ca-activated K channels in rat myotubule,38 bovine chromaffin cells,39 and human cultured blood monocytes.40 Although Ca-activated K channels with small conductances of 10 to 19 pS have been reported in erythrocytes,4’ snail neurons4* and rat skeletal muscles,43the evaluation of the similarity of these channels to those found here needs further investigation. These two types of channels probably mediate the Ca-activated K permeability change in human alveolar macrophages stimulated with ZAS and Ca ionophore A23 187, such as we used for observing chemiluminescence response or LTB, release. Quinine has been reported to inhibit Ca-activated K conductance change in mouse macrophages,” which agrees with our findings that quinine reduced both types of Ca-activated K channel currents in human alveolar macrophages (Fig. 4). We observed that quinine inhibited chemiluminescence response and LTB, release induced by ZAS or Ca ionophore (Figs. 5 and 6). It appears that quinine inhibits some mechanism(s) that induce chemiluminescence and LTB4 release. Quinine reduced Caactivated K channel currents in human alveolar macrophages. Therefore, the inhibitory effect of quinine on mediator release may be closely related to blocking of K channels, and the increase in K concentration near extracellular surface of the membrane caused by the activation of K channels or conformational change of K channel protein, etc., may affect the mechanism(s) that cause chemiluminescence and LTB, release. Our preliminary observation of reduction of PMA-induced chemiluminescence by valinomycinz4 (a K ionophore) may reflect close association of K channels with the activational response of macrophages. When the molecular mechanism of the interaction of Ca-activated K channels (or K currents) with the cellular response of alveolar macrophages become more clear, detailed examination of the relationships between the channel currents and the cellular response will be possible. We observed that quinine also inhibited chemiluminescence responses induced by PMA. PMA has been reported to induce its biologic responses through persistent activation of protein kinase C,44 without increase in cytosolic Ca levels.45 Moreover, in mouse peritoneal macrophages, the application of PMA

ti channels

VOLUME Xl NUMB% ?

causes depolarization,4 which precedes a detectable release of superoxide anion. Therefore, the behavior of Ca-activated K channels of macrophages stimulated with PMA may be different from those activated by endotoxin activated serum N-formyl-methionylleucyl-phenylalanine, and Ca ionophore, which cause hyperpolarization. 14.” Although further studies must be done. incomplete activation of small channels by [Cal, (Fig. 3) may be related to this problem (via protein kinase C etc). How PMA acts on channel currents in human alveolar macrophages needs further investigation. Lucigenin-dependent chemiluminescence is probably related to the production of superoxide anion4’ that affects the permeability of epithelial cel1s.j LTB, is a potent chemotactic agent for polymorphonuclear cells, including neutrophils and eosinophils.” These cells have been reported to be closely related to asthmatlc responses, such as ozone-induced hyperreactivity,‘Y late asthmatic response,” and epithelial damage.” Therefore, the inhibition of the alveolar macrophage through blockade of Ca-activated K channels may be meaningful in the treatment of asthma. Further investigation will elucidate the role of K conductance changes in alveolar macrophages, both the normal and asthmatic states. We arc indebted to Drs. M. Blennerhassettand R. Scott for critical reading of the manuscript. Specialthanks to Drs. J. Btenenstock and J. M. Drazen for their useful and Mrs. J. Butera for typing the manuscript.

comments

REFERENCES I. loseph M, Capron A. IgE receptors on macrophages: biological significance. Agents Actions 1985;16:27. 2. loseph M, Tonne1 AB, Capron A, Voisin C. Enzyme release and superoxide anion production by human alveolar macrophages stimulated with immunoglobulin E. Clin Exp Immunol 1980;40:416. 3. Welsh MJ, Shasby DM, Husted RM. Oxidants increase paracellular permeability in a cultured epithelial cell line. J Clin Invest 1985;76:1155. 4. Schleimer RP, Fox CC, Naclerio RM, Plaut M, Creticos PS, Togias AG, Warner JA, Kagey-Sobotka A, Lichtenstein LM. Role of human basophils and mast cells in the pathogenesis of allergic diseases. J ALLERGY CLIN IMMUNOL 1985;76:369. 5. Golden JA, Nadel JA, Boushey HA. Bronchial hyperirritability in healthy subjects after exposure to ozone. Am Rev Respir Dis ) 978:118:287. 6. Morey J, Page CP, Sanjar S. The platelet in asthma. Lancet 1984;2:1142. 7. Rankin JA, Hitchcock M, Merrill WW, Huang SS, Brasler JR, Bach MK, Askenase PW. IgE immune complexes induce immediate and prolonged release of leukotriene C, (LTC,) from rat alveolar macrophages. J Immunol 1984;132:1993. 8. Denjean A. Amoux B. Masse R, Lockhart A, Benveniste J. Acute effects of intratracheal administration of plateletactivating factor in baboons. J Appl Physiol 1983;55:799.

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macrnl:haqes

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9. Holmyde MC, Altounyan REC, Coie M. l)~xct:i M, l3111trt I!\‘. Bronchoconstriction produced in man bv I~ul\cl:~ic~c r ar.d 1) Lancet 1981:2:17. 10. Gosset P, Tonne1 AB, Joseph M, Prin L Ualldn A. Charon J, Capron A. Secretion of a chcmot,~cti~~ factor tor neutrophils and eosinophils by alvcoiar ma< rophages rrom asthmatic patients. J ALLERGY CLIMB IUML:NOI :981.74.XZ7 1 I. de Monchy JGR, Kauffman HF, Venge P. Kseter GH, Janscn HM, Sluiter HJ. de Vries K. Bronchoaiveolnr cosinophitia during allergen-induced late asthmatIc reactit>rr %m Rrr\- Re+ pir Dis 1985;131:373. 12. Frigas E, Loegering DA. Solley CO, Farrow a.j.M. Gle~h GJ. Elevated levels of the eosinophil granule maj~ basic protein in the sputum of patients with bronchial :ISI~“I~ Ma;,,> (‘itn Proc 1981:56:345. 13. Schulman ES, Liu MC. Proud D. MacGiashan JI DU. i.lchtenstein LM. Plaut M. Human lung macrophares induce Inatamine release from basophils and tn:Ist ceil& ‘$1~1Kc\ Kespir Dis 1985;131:230. 14. Gallin EK. Wiederhold ML, LIpsky PE. Koserrrhal Al. Spontaneous and induced membrane hyperpolarv,&x~~ in macrophagea. J Cell Physiol 1975;86:653. 15. Gallin EK. Gallin Jl. Interaction ot chemotaciic factnr~ with human macrophages. J Cell Biol 1977:75:?’ 16. Gallin EK. Electrophysiological propenrcs ill rnacrophapes. Fed Proc 1984;43:2385. 17. Kitagawa S. Johnston RB Jr. Deaclivallon ol the resprratory burst in activated macrophages: evidence for altcratmn ot sIgna transduction. J lmmunol 1986; l36:2605. 18. Hamill OP. Marty A. Nehr E. Sackmann B. S&worth FJ Improved patch clamp techniques for high-re*olution current recording from cells and cell-free membrane rr:ttctre\. PRiigers Arch 1981;391:85. 19. DOS Reis G, Pereschini P, Ribeiro J. Ohvcira-Castro G. Electrophysiology of phagocytic membranes. II. Membrane ptential and induction of slow hyperpolarizattan~~ m wee macrophages. Biochim Biophys Acta 197’r:55?.3i I 20. Holian A, Damele RP. The role 01. calcium in the imllatlon of superoxide release from alveolar macrophaar .! Cell Physiol 1982;i 13:87. 21. Schopf RE. Mattar J. Meyenburg %‘. Schem:r 0, Hammann KP, Lemmel EM. Measurement of the respiratory burst in human monocytes and polymorphonuclear leukocytes by nitro blue tetrazolium reduction and chem~luminezzcncc:. J Immunol Methods 1984;67:109. 22. Lee TH, Drazen JM, Lewis RA. Austen Kt, Substrate and regulatory functions of eicosapentaenoic and ducosahexaenoic acids for the S-lipoxygenase parhwrrt Prop Biochem Pharmacol 1985;20: 1. 23. Kakuta Y, Kanno T, Ohyama T, Sasakl H, l‘ab&moaT Single Ca-activated K channels in human alveolar macrophages. Am Rev Respir Dis 1985;13l:A390. 24. Kdkuta Y. Okayama H, Aikawa T, Kanrr*i ‘I‘. Saaaki H. Kato T, Takishima T. Involvement of K in i:cllolar responses of human alveolar macrophages. Am Re* Kcspir Die 19X7: 135:A345. 25. Tomioka M. Ida S, Shindoh Y, lshrhara ‘T, ‘I’akish~ma T. Mast cells in bronchoalveolar lumen of patients with bronchial asthma. Am Rev Respir Dis 1984; 129: 1000 26. Kakuta Y, Kato T, Sasaki H. Takishima T Effect of ketotlfen on human alveolar macrophages. J AI I.ER( b C‘I IV I~%.~~I;NoI 1988;81:469-74. 27. Saltini C, Hance AJ. Ferrans VJ, Basset j-. Bitterman PB, Crystal RG. Accurate quantification of cells recovered by bronchoalveolar lavage Am Rev Respir I& l%U:l3!!-6%~

468

Kakuta

et al.

28. Barrett JN, Magleby KL, Pallotta BS. Properties of single calcium-activated potassium channels in cultured rat muscle. J Physiol 1982;331:211. 29. Pallotta BS. Calcium-activated potassium channels in rat muscle inactivate from a short-duration open state. J Physiol 1985;363:501. 30. Kakuta Y. Effects of ATP and related compounds on the Cainduced Ca release mechanism of the Xenopus SR. Pfliiger Arch 1984;400:72. 3 1. Kato T, Terui T, Tagami H. Chemotactic factor-induced chemiluminescence of human neutrophils. In: Hayashi 0, Imamura S, Miyachi Y, eds. The biological role of reactive oxygen species in skin. Tokyo: University of Tokyo Press, 1986: 171-8. 32. Hoidal JR, Repine JE, Beall CD, Rasp FL Jr, White JG. The effect of phorbol myristate acetate on the metabolism and ultrastmcture of human alveolar macrophages. Am J Path01 1978;91:469. 33. Williams JD, Czop JK, Austen KF. Release of leukotrienes by human monocytes on stimulation of their phagocytic receptor for particulate activators. J Immunol 1984;132:3034. 34. Lowrey OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265. 35. Latorre R, Miller C. Conduction and selectivity in potassium channels. J Membr Biol 1983;71:11. 36. Hartshome RP, Keller BU, Talvenheimo JA, Catterall WA, Montal M. Functional reconstitution of the purified brain sodium channel in planar lipid bilayers. Proc Nat1 Acad Sci USA 1985;82:240. 37. Fels AO, Pawlowski NA, Cramer EB, King TKC, Cohn ZA, Scott WA. Human alveolar macrophages produce leukotriene B,. Proc Nat1 Acad Sci USA 1982;79:7866. 38. Pallotta BS, Magleby KL, Barrett JN. Single channel record-

J. ALLERGY CLIN. IMMUNOL. FEBRUARY 1988

ings of Ca2’- activated K+ currents in rat muscle cell culture. Nature 1981;293:471. 39. Marty A. Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature 198 1;29 I :497. 40. Gallin EK. Calcium- and voltage-activated potassium channels in human macrophages. Biophys J 1984;46:821. 41. Hamill OP. Potassium channel currents in human red blood cells. J Physiol 1981;319:97P. 42. Lux HD, Neher E, Marty A. Single channel activity associated with the calcium-dependent outward current in Helixpomatia. Pfliigers Arch 1981;389:293. 43. Blatz AL, Magleby KL. Single apamin-blocked Ca-activated K+ channels of small conductance inn cultured rat skeletal muscle. Nature 1986;323:718. 44. Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science 1984;225: 1365. 45. Di Virgilio F, Lew DP, Pozzan T. Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca levels. Nature 1984;310:691. 46. Kitagawa S, Johnston RB Jr. Relationship between membrane potential changes and superoxide-releasing capacity in resident and activated mouse peritoneal macrophages. J Immunol 1985;135:3417. 47. Williams AJ, Cole PJ. Investigation of alveolar macrophage function using lucigenin-dependent chemiluminescence. Thorax 1981;36:866. 48. O’Byme PM, Waters EH, Gold BD, Aizawa HA, Fabby LM, Alpert SE, Nadel JA, Holtzman MJ. Neutrophil depletion inhibits airway hyperresponsiveness induced by ozone exposure in dogs. Am Rev Respir Dis 1984;130:214. 49. Hodgkin AL, Katz B. The effect of sodium ions on the electrical activity of the giant axon of the squid. J Physiol 1949:108:37.

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